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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (2): 224-236.doi: 10.3724/SP.J.1006.2021.01042


TaPP2-A13 gene shows induced expression pattern in wheat responses to stresses and interacts with adaptor protein SKP1 from SCF complex

MENG Yu-Yu1(), WEI Chun-Ru1, FAN Run-Qiao1, YU Xiu-Mei1,2,*(), WANG Xiao-Dong2, ZHAO Wei-Quan2, WEI Xin-Yan2, KANG Zhen-Sheng3, LIU Da-Qun2   

  1. 1College of Life Sciences, Hebei Agricultural University / Hebei Key Laboratory of Plant Physiology and Molecular Pathology, Baoding 071001, Hebei, China
    2Technological Innovation Center for Biological Control of Crop Diseases and Insect Pests of Hebei Province, Baoding 071001, Hebei, China
    3College of Plant Protection, Northwest A&F University / State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, Shaanxi, China
  • Received:2020-05-12 Accepted:2020-09-13 Online:2021-02-12 Published:2020-09-30
  • Contact: YU Xiu-Mei E-mail:183656991@qq.com;yuxiumeizy@126.com
  • Supported by:
    Higher Education Science and Technology Research Project of Hebei Province(ZD2019086);Natural Science Foundation of Hebei Province(C2020204050);National Natural Science Foundation of China(31301649);Open Research Fund of State Key Laboratory of Crop Stress Biology for Arid Areas(CSBAAKF2018008)


To explore the function and molecular mechanism of Phloem protein 2 (PP2) gene in wheat (Triticum aestivum L.) response to stresses, a TaPP2-A13 putatively encoding a PP2 protein was obtained from TcLr15, a wheat near isogenic line against leaf rust pathogen, in the present study. The complete coding region of TaPP2-A13 encodes a hydrophilic polypeptide with molecular weight of 33.18 kD, and theoretical isoelectric point is 6.36. There is an F-box domain at N-terminal and a PP2 domain at C-terminal of the TaPP2-A13 protein sequence, which indicates that wheat TaPP2-A13 belongs to F-box/PP2 (FBP) subfamily. Wheat TaPP2-A13 shared relatively higher sequence similarity with PP2-A13 from Gramineae. Quantitative real-time PCR (qRT-PCR) results indicated that TaPP2-A13 was induced by infection of leaf rust pathogen (Puccinia triticina), and showed stronger expression in susceptible combination than that in resistant one. An obvious up-regulation of TaPP2-A13 was observed after treatment with abscisic acid (ABA), salicylic acid (SA) and methyl jasmonate (MeJA) in wheat. TaPP2-A13 was significantly down-regulated after treatment with PEG and H2O2, while TaPP2-A13 striking increased first, then fell down after NaCl treatment in wheat. Subcellular localization result indicated that TaPP2-A13 distributed in both of the nucleus and cytoplasm. The recombinant vector BD-TaPP2-A13 was used as the bait to screen Yeast 2 Hybrid (Y2H) library, 11 kinds of proteins were finally obtained. Further Y2H assays identified that TaPP2-A13 physically interacted with five kinds of proteins including TaPP2C5, TaSLY1, TaCHI, TaRbcS, and TaSKP1. BiFC and Co-IP results further confirmed that TaPP2-A13 interacted with TaSKP1, an adaptor protein from SKP1-Cullin-F-box (SCF) complex, which made us to speculate that TaPP2-A13 functions as a member of SCF complex by binding with TaSKP1. These findings laid some foundation for further analyzing the function of TaPP2-A13 and exploring its regulatory network.

Key words: phloem protein 2, wheat leaf rust, abiotic stresses, hormone, expression pattern, protein interaction

Table 1

Primer information used in this study"

Primer name
Forward primer sequence
Reverse primer sequence
temperature (℃)
TaPP2-A13-His-F/R gctgatatcggatccgaattcATGGGGGC GGGGGCTTCG tgcggccgcaagcttgtcgacTTACTTGCGTATGC ACTCCTCG 60.0
TaPP2-A13-GFP-F/R atacaccaaatcgactctagaATGGGGGC GGGGGCTTCG catggtaccggatccactagtCTTGCGTATGCACT CCTCGG 60.0
TaPP2-A13-NE-F/R cccaggcctactagtggatccATGGGGGC GGGGGCTTCG agcggtaccctcgaggtcgacCTTGCGTATGCACT CCTCGG 60.0
TaSKP1-FLAG-F/R gagaacacgggggactctagaATGGCGG CCGCGGGAGAC cgtcctaggcttaagtctagaCTCAAAGGCCCACT GGTTCTC 60.0
TaPP2-A13-HA-F/R agacttaagcctaggacgcgtATGGGGGC GGGGGCTTCG AtcgtatgggtacatacgcgtCTTGCGTATGCACT CCTCGG 60.0

Fig. 1

Alignment for amino acid sequence between TaPP2-A13 and other PP2-A13 from gramineous plants Region with black box represents F-box domain, region with gray frames represents PP2 domain; plant species used in this study are Triticum aestivum, Aegilops tauschii, Brachypodium distachyon, Dichanthelium oligosanthes, Oryza brachyantha, Oryza sativa, Panicum hallii, Panicum miliaceum, Setaria italica, Setaria viridis, and Zea mays."

Fig. 2

Phylogenetic tree of plant PP2-A13 Solid line outside of the phylogenetic tree: the clade of plant PP2-A13 proteins; ●: TaPP2-A13 protein; Plant species used are Apostasia shenzhenica, Phalaenopsis equeatris, Dendrobium catenatum, Phoenix dactylifera, Elaeis guineensis, Ensete ventricosum, Musa balbisiana, Musa acuminate, Papaver somniferum, Macleaya cordata, Rhodamnia argentea, Medicago truncatula, Jatropha curcas, Salix brachista, Populus euphratica, Ananas comosus, Triticum urartu, Eragrostis curvula, Oryza brachyantha, Oryza sativa japonica Group, Oryza sativa indica Group, Zea mays, Sorghum bicolor, Setaria viridis, Setaria italica, Dichanthelium oligosanthes, Panicum miliaceum, Panicum hallii, Brachypodium distachyon, Hordeum vulgare, Triticum aestivum, and Aegilops tauschii."

Fig. 3

Expression patterns of TaPP2-A13 gene under different exogenous hormones treatment in wheat TaGAPDH served as a reference gene. * indicates signi?cant differences at P < 0.05 in samples at different treatment time points compared to the sample at 0 hour. Error bars represent the standard error of each sample (n = 3)."

Fig. 4

Expression patterns of TaPP2-A13 gene under different biotic/abiotic stresses in wheat TaGAPDH served as a reference gene. * indicates signi?cant differences at P < 0.05 in samples at different treatment time points compared to the sample at 0 hour. Error bars represent the standard error of each sample (n = 3)."

Fig. 5

Subcellular localization of TaPP2-A13-GFP protein The green fluorescence under fluorescence microscope represents GFP protein. Bar = 100 μm."

Table 2

Results of library screening by TaPP2-A13"

Accession No.
Protein name
Number of clones
XP_020153404.1 Protein phosphatase 2C 5 (PP2C5) 1
KU857044.1 S-phase kinase-associated protein 1 (SKP1) 1
XM_020332687.1 UDP-glucose flavonoid 3-O-glucosyl transferase 7-like 3
EMS45698.1 SEC1 family transport protein SLY1 1
XM_020338494.1 3-ketoacyl-CoA thiolase 2, peroxisomal-like 2
LM992844.1 Rca1_beta gene for RUBISCO activase beta (TaRca1_beta) 3
KC776912.1 Ribulose activase A 2
AK330458.1 Ribulose-bisphosphate carboxylase small chain (RbcS) 1
AF251264.1 Ribulose bisphosphate carboxylase activase B (RcaB) 3
AY123222.1 Putative Fe-S precursor protein 2
BAB82472.1 Chitinase 2 1

Fig. 6

Interaction of TaPP2-A13 and screening proteins identified by Y2H"

Fig. 7

Interaction of TaPP2-A13 and TaSKP1 identified by BiFC The green fluorescence under fluorescence microscope represents GFP protein. Bar = 100 μm."

Fig. 8

Interaction of TaPP2-A13 and TaSKP1 in Nicotiana benthamiana leaves by Co-IP Total protein extracts of N. benthamiana were incubated with anti-HA affinity beads, the immunoprecipitates were analyzed by immunoblotting with anti-FLAG and anti-HA antibodies."

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